RESUMEN
Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Roturas del ADN de Cadena Simple , Proteína Homóloga de MRE11/deficiencia , Neocórtex/metabolismo , Poli ADP Ribosilación , Proteostasis , Ataxias Espinocerebelosas/metabolismo , Adulto , Línea Celular Tumoral , Femenino , Humanos , Masculino , Neocórtex/patología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patologíaRESUMEN
BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.
Asunto(s)
Proteína BRCA2/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteína de Replicación A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Daño del ADN/efectos de los fármacos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/efectos de los fármacos , Humanos , Hidroxiurea/farmacología , Proteína Homóloga de MRE11/deficiencia , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Mutación , Fosforilación , ARN Interferente Pequeño , Recombinasa Rad51/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
AIM: To evaluate the frequency of loss of mediator of DNA damage checkpoint protein 1 (MDC1) protein expression in endometrial cancer (EC) and to determine whether loss of MDC1 is associated with certain clinicopathological parameters. MATERIALS AND METHODS: MDC1 expression was examined in 426 samples of EC. The nuclear immunoreactivity of the protein was defined as: negative, weak, moderate and strong. RESULTS: Loss of MDC1 expression (defined as negative nuclear staining) was found in 8.9% (38/426) of ECs and was significantly associated with the loss of MRE11 homolog, double-strand break repair nuclease, RAD50 double-strand break repair protein and nibrin complex components. In addition, loss of expression of MDC1 showed a significant correlation with any mismatch repair deficiency, with endometrioid histological subtype and low tumour grading. CONCLUSION: Based on these findings, we suggest that MDC1 loss frequently occurs in ECs with microsatellite instability. Due to deficient homologous recombination DNA repair, MDC1-negative ECs might show an increased sensitivity to poly(ADP-ribose) polymerase-inhibitory therapy.
